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Biofilm formation on endotracheal tubes (ETT) is an important factor in the development of ventilator-associated pneumonia (VAP). This work aimed to investigate the effectiveness of colistin (COL) against the early stages of biofilm formation by Pseudomonas aeruginosa. Two strategies were used: pre-conditioning the adhesion surfaces with COL before biofilm formation and growing biofilms in its presence. The combined effect of treating P. aeruginosa 24-hours old biofilms with Ciprofloxacin (CIP) or colistin (COL) on clean and COL-conditioned surfaces was also assessed. Random deposition of COL residues altered the physico-chemical properties of the adhesion surfaces and impaired biofilm formation. Moreover, as a consequence of the reduced amount of biofilms attached to COL conditioned surfaces, adhered cells became more exposed to the subsequent action of CIP or COL, suggesting a combined outcome of prophylactic and therapeutic COL-based strategies. Results highlighted the promising use of COL to prevent the establishment of biofilms on ETT., This work was supported by the Portuguese Foundation for Science Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit BioTecNorte operation [NORTE-01-0145-FEDER-000004] funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte. The authors also acknowledge COMPETE2020 FCT for the project POCI-01-0145-FEDER-029841 and for PhD Grant of Diana Alves (SFRH/BD/78063/2011)., info:eu-repo/semantics/publishedVersion

The endotracheal tube (ETT) affords support for intubated patients, but the rising incidence of ventilator-associated pneumonia (VAP) is jeopardizing its application. ETT surfaces promote (poly)microbial colonization and biofilm formation, with a heavy burden for VAP. Devising safe, broad-spectrum antimicrobial materials to tackle ETT bioburden is needful. Herein, we immobilized ciprofloxacin (CIP) and/or chlorhexidine (CHX), through polydopamine (pDA)-based functionalization, onto polyvinyl chloride (PVC) surfaces. These surfaces were characterized and challenged with single and polymicrobial cultures of VAP-relevant bacteria (Pseudomonas aeruginosa;Acinetobacter baumannii;Klebsiella pneumoniae;Staphylococcus aureus;Staphylococcus epidermidis) and fungi (Candida albicans). The coatings imparted PVC surfaces with homogeneous morphology, varied wettability, and low roughness. Coated surfaces exhibited sustained CIP/CHX release, retaining long-term (10 days) stability. Surfaces evidencing no A549 lung cell toxicity exhibited broad-spectrum anti-biofilm activity. CIP/CHX co-immobilization resulted in better outcomes than CIP or CHX coatings, reducing bacteria up to >7 Log10, and modestly distressing (ca. 1 Log10)C. albicans. The anti-biofilm effectiveness of coated surfaces endured for dual biofilms, substantially preventing bacterial populations and fungi (ca. 2.7 Log10) inP. aeruginosa/C. albicansconsortia. A less pronounced antifungal effect (ca. 1 Log10reduction) was found in triple-species communities, but fully preventingP. aeruginosaandS. aureuspopulations. CIP/CHX co-immobilization holds a safe and robust broad-spectrum antimicrobial coating for PVC-ETTs, with the promise laying in reducing VAP incidence.

Aim: To investigate the role of pre-established Staphylococcus aureus on Pseudomonas aeruginosa adaptation and antibiotic tolerance. Materials & methods: Bacteria were cultured mimicking the sequential pattern of lung colonization and exposure to ciprofloxacin. Results: In the absence of ciprofloxacin exposure, S. aureus and P. aeruginosa coexisted supported by the physicochemical characteristics of the artificial sputum medium. S. aureus had no role in P. aeruginosa tolerance against ciprofloxacin and did not select P. aeruginosa small-colony variants during antibiotic treatment. rhlR and psqE were downregulated after the contact with S. aureus indicating that P. aeruginosa attenuated its virulence potential. Conclusion: P. aeruginosa and S. aureus can cohabit in cystic fibrosis airway environment for long-term without significant impact on P. aeruginosa adaptation and antibiotic tolerance.

The main goal of this study was to chemically characterize an aqueous S. nigra flower extract and validate it as a bioactive agent. The elderflower aqueous extraction was performed at different temperatures (50, 70 and 90 °C). The extract obtained at 90 °C exhibited the highest phenolic content and antiradical activity. Therefore, this extract was analyzed by GC-MS and HPLC-MS, which allowed the identification of 46 compounds, being quercetin and chlorogenic acid derivatives representative of 86% of the total of phenolic compounds identified in hydrophilic fraction of the aqueous extract. Naringenin (27.2%) was the major compound present in the lipophilic fraction. The antiproliferative effects of the S. nigra extract were evaluated using the colon cancer cell lines RKO, HCT-116, Caco-2 and the extracts antigenotoxic potential was evaluated by the Comet assay in RKO cells. The RKO cells were the most susceptible to S. nigra flower extract (IC50=1250 µg mL1). Moreover, the extract showed antimicrobial activity against Gram-positive bacteria, particularly Staphylococcus aureus and S. epidermidis. These results show that S. nigra-based extracts can be an important dietary source of bioactive phenolic compounds that contribute to health-span improving life quality, demonstrating their potential as nutraceutical, functional foods and/or cosmetic components for therapeutic purposes., This research was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and for Scientific Employment Stimulus 2017 (CEECIND/01507/2017) provided to Ana M. Sousa., info:eu-repo/semantics/publishedVersion

Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-50094-3., While considerable research has focused on studying individual-species, we now face the challenge of determining how interspecies interactions alter bacterial behaviours and pathogenesis. Pseudomonas aeruginosa and Staphylococcus aureus are often found to co-infect cystic-fibrosis patients. Curiously, their interaction is reported as competitive under laboratory conditions. Selecting appropriate methodologies is therefore critical to analyse multi-species communities. Herein, we demonstrated the major biases associated with qPCR quantification of bacterial populations and optimized a RNA-based qPCR able not only to quantify but also to characterize microbial interactions within dual-species biofilms composed by P. aeruginosa and S. aureus, as assessed by gene expression quantification. qPCR quantification was compared with flow-cytometry and culture-based quantification. Discrepancies between culture independent and culture dependent methods could be the result of the presence of viable but not-cultivable bacteria within the biofilm. Fluorescence microscopy confirmed this. A higher sensitivity to detect viable cells further highlights the potentialities of qPCR approach to quantify biofilm communities. By using bacterial RNA and an exogenous mRNA control, it was also possible to characterize bacterial transcriptomic profile, being this a major advantage of this method., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Grant of APM (SFRH/BD/132165/2017)., info:eu-repo/semantics/publishedVersion

Growing antimicrobial resistance and biofilm infection resilience have led researchers to study the potential laying in antimicrobial combinations, including those encompassing enzymes with biofilm disrupting abilities. Therefore, this work set out to evaluate the undergone journey of antimicrobial enzyme combination research and gain insights into its current status and most promising leads. Expert curators annotated and analysed all published experimental data on enzyme-encompassing combinations for two major biofilm-forming pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. This entailed the construction of the first publically accessible online database on antimicrobial enzyme combinations, the Antimicrobial Enzyme Combinations Database (www.ceb.uminho.pt/aecd). Gathered data was also reconstructed as knowledge-networks to help analyse and visualize annotated entities (e.g. enzymes, methods, strains, combination outputs). The database currently holds 122 and 206 annotated combinations for P. aeruginosa and S. aureus, respectively, and their analysis allowed a systematic review of the available evidence on enzyme combinations, reliably illustrating the studies being performed. The most tested enzymes (e.g. lysozyme, DNAse, lysostaphin) were scrutinised and the rationale behind each combination explained. This research area is still growing even though current research gaps/opportunities were identified, such as lack of biofilm testing and of studies on polymicrobial scenarios. Hopefully, this work will shed light on the synergetic potential resting in enzyme combinations and alleviate some of the time and resource-consuming tasks related to enzyme combination research by helping the selection and design of new enzyme related therapeutic options for P. aeruginosa and S. aureus infections., This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 [POCI-01-0145-FEDER-006684] and BioTecNorte operation [NORTE-01-0145-FEDER-000004], funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors also recognise COMPETE2020 and FCT for the project POCI-01-0145-FEDER-029841., info:eu-repo/semantics/publishedVersion

Accepted Article, Summary Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent respiratory pathogens in cystic fibrosis (CF) patients. Both organisms often cause chronic and recalcitrant infections, in large part due to their ability to form biofilms, being these mixed-species infections correlated with poor clinical outcomes. In this study, the hypothesis that S. aureus adopts phenotypes allowing its coexistence with P. aeruginosa during biofilm growth was put forward. We noticed that S. aureus undergoes a viable but non-cultivable (VBNC) state in the dominated P. aeruginosa dual-species consortia, whatsoever the strains used to form the biofilms. Moreover, an increased expression of genes associated with S. aureus virulence was detected suggesting that the phenotypic switching to VBNC state might account for S. aureus pathogenicity and, in turn, influence the clinical outcome of the mixed-species infection. Thus, P. aeruginosa seems to induce both phenotypic and transcriptomic changes in S. aureus, helping its survival and co-existence in the dual-species biofilms. Overall, our findings illustrate how interspecies interactions can modulate bacterial virulence in vitro, contributing to a better understanding of the behaviour of P. aeruginosa-S. aureus dual-species biofilms. This article is protected by copyright. All rights reserved., Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte. The authors also acknowledge the support, through the Programa Operacional Competitividade e Internacionalização (COMPETE2020) and by national funds, through the Portuguese Foundation for Science and Technology (FCT), of the POLY-PrevEnTT project (PTDC/BTMSAL/29841/2017-POCI-01-0145-FEDER-029841), and for the Scientific Employment Stimulus 2017 (CEECIND/01507/2017), info:eu-repo/semantics/publishedVersion

Antimicrobial therapy is facing a worrisome and underappreciated challenge, the phenomenon of heteroresistance (HR). HR has been gradually documented in clinically relevant pathogens (e.g. Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia spp., Acinetobacter baumannii, Klebsiella pneumoniae, Candida spp.) towards several drugs and is believed to complicate the clinical picture of chronic infections. This type of infections are typically mediated by polymicrobial biofilms, wherein microorganisms inherently display a wide range of physiological states, distinct metabolic pathways, diverging refractory levels of stress responses, and a complex network of chemical signals exchange. This review aims to provide an overview on the relevance, prevalence, and implications of HR in clinical settings. Firstly, related terminologies (e.g. resistance, tolerance, persistence), sometimes misunderstood and overlapped, were clarified. Factors generating misleading HR definitions were also uncovered. Secondly, the recent HR incidences reported in clinically relevant pathogens towards different antimicrobials were annotated. The potential mechanisms underlying such occurrences were further elucidated. Finally, the link between HR and biofilms was discussed. The focus was to recognize the presence of heterogeneous levels of resistance within most biofilms, as well as the relevance of polymicrobial biofilms in chronic infectious diseases and their role in resistance spreading. These topics were subject of a critical appraisal, gaining insights into the ascending clinical implications of HR in antimicrobial resistance spreading, which could ultimately help designing effective therapeutic options., This work was supported by the Portuguese Foundation for Science Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit BioTecNorte operation [NORTE-01-0145-FEDER-000004] funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte. The authors also acknowledge COMPETE2020 FCT for the project POCI-01-0145-FEDER-029,841 and for the Scientific Employment Stimulus 2017 grant [CEECIND/01507/2017] (A. M. Sousa)., info:eu-repo/semantics/publishedVersion

The Supplementary Material for this article canbe found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2020.550505/ full#supplementary-material, Modern medicine is currently facing huge setbacks concerning infection therapeutics as microorganisms are consistently knocking down every antimicrobial wall set before them. The situation becomes more worrying when taking into account that, in both environmental and disease scenarios, microorganisms present themselves as biofilm communities that are often polymicrobial. This comprises a competitive advantage, with interactions between different species altering host responses, antimicrobial effectiveness, microbial pathogenesis and virulence, usually augmenting the severity of the infection and contributing for the recalcitrance towards conventional therapy. Pseudomonas aeruginosa and Candida albicans are two opportunistic pathogens often co-isolated from infections, mainly from mucosal tissues like the lung. Despite the billions of years of co-existence, this pair of microorganisms is a great example on how little is known about cross-kingdom interactions, particularly within the context of coinfections. Given the described scenario, this study aimed to collect, curate, and analyze all published experimental information on the molecular basis of P. aeruginosa and C. albicans interactions in biofilms, in order to shed light into key mechanisms that may affect infection prognosis, increasing this area of knowledge. Publications were optimally retrieved from PubMed and Web of Science and classified as to their relevance. Data was then systematically and manually curated, analyzed, and further reconstructed as networks. A total of 641 interactions between the two pathogens were annotated, outputting knowledge on important molecular players affecting key virulence mechanisms, such as hyphal growth, and related genes and proteins, constituting potential therapeutic targets for infections related to these bacterial-fungal consortia. Contrasting interactions were also analyzed, and quorum-sensing inhibition approaches were highlighted. All annotated data was made publicly available at www.ceb.uminho.pt/ISCTD, a database already containing similar data for P. aeruginosa and Staphylococcus aureus communication. This will allow researchers to cut on time and effort when studying this particular subject, facilitating the understanding of the basis of the inter-species and inter-kingdom interactions and how it can be modulated to help design alternative and more effective tailored therapies. Finally, data deposition will serve as base for future dataset integration, whose analysis will hopefully give insights into communications in more complex and varied biofilm communities., This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte. The authors also acknowledge COMPETE2020 and FCT for the project POCI-01-0145-FEDER-029841 and FCT for the PhD Grant of TG [grant number SFRH/BD/136544/2018]., info:eu-repo/semantics/publishedVersion

A crucial factor in the pathogenesis of orthopedics associated infections is that bacteria do not only colonize the implant surface but also the surrounding tissues. This study aimed to engineer an antimicrobial release coating for stainless steel (SS) surfaces, to impart them with the ability to prevent Staphylococci colonization. Chlorhexidine (CHX) was immobilized using two polydopamine (pDA)-based approaches: a one-pot synthesis, where CHX is dissolved together with dopamine before its polymerization; and a two-step methodology, comprising the deposition of a pDA layer to which CHX is immobilized. To modulate CHX release, an additional layer of pDA was also added for both strategies. Immobilization of CHX using a one-step approach yielded surfaces with a more homogenous coating and less roughness than the other strategies. The amount of released CHX was lower for the one-step approach, as opposed to the two-step approach yielding the higher release, which could be decreased by applying an outward layer of pDA. Both one and two-step approaches provided the surfaces with the ability to prevent bacterial colonization of the surface itself and kill most of bacteria in the bulk phase up to 10 days. This long-term antimicrobial performance alluded a stable and enduring immobilization of CHX. In terms of biocompatibility, the amount of CHX released from the one-step approach did not compromise the growth of mammalian cells, contrary to the two-step strategy. Additionally, the few bacteria that managed to adhere to surfaces modified with one-step approach did not show evidence of resistance towards CHX. Overall data underline that one-step immobilization of CHX holds great potential to be further applied in the fight against orthopedic devices associated infections., This study was supported by the Portuguese Foundation for Science and Tecnhnology (FCT) under the scope of the strategic funding of UID/ BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145- FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte. The authors also acknowledge the support, through the Programa Operacional Competitividade e Internacionalizaçao ˜ (COMPETE2020) and by national funds, through the FCT, of the POLY-PrevEnTT project (PTDC/BTMSAL/29841/2017-POCI-01-0145-FEDER-029841) and the PhD Grant of Tania ˆ Grainha (SFRH/BD/136544/2018)., info:eu-repo/semantics/publishedVersion

Modern medicine is currently facing huge setbacks concerning infection therapeutics as microorganisms are consistently knocking down every antimicrobial wall set before them. The situation becomes more worrying when taking into account that, in both environmental and disease scenarios, microorganisms present themselves as biofilm communities that are often polymicrobial. This comprises a competitive advantage, with interactions between different species altering host responses, antimicrobial effectiveness, microbial pathogenesis and virulence, usually augmenting the severity of the infection and contributing for the recalcitrance towards conventional therapy. Pseudomonas aeruginosa and Candida albicans are two opportunistic pathogens often co-isolated from infections, mainly from mucosal tissues like the lung. Despite the billions of years of co-existence, this pair of microorganisms is a great example on how little is known about cross-kingdom interactions, particularly within the context of coinfections. Given the described scenario, this study aimed to collect, curate, and analyze all published experimental information on the molecular basis of P. aeruginosa and C. albicans interactions in biofilms, in order to shed light into key mechanisms that may affect infection prognosis, increasing this area of knowledge. Publications were optimally retrieved from PubMed and Web of Science and classified as to their relevance. Data was then systematically and manually curated, analyzed, and further reconstructed as networks. A total of 641 interactions between the two pathogens were annotated, outputting knowledge on important molecular players affecting key virulence mechanisms, such as hyphal growth, and related genes and proteins, constituting potential therapeutic targets for infections related to these bacterial-fungal consortia. Contrasting interactions were also analyzed, and quorum-sensing inhibition approaches were highlighted. All annotated data was made publicly available at www.ceb.uminho.pt/ISCTD, a database already containing similar data for P. aeruginosa and Staphylococcus aureus communication. This will allow researchers to cut on time and effort when studying this particular subject, facilitating the understanding of the basis of the inter-species and inter-kingdom interactions and how it can be modulated to help design alternative and more effective tailored therapies. Finally, data deposition will serve as base for future dataset integration, whose analysis will hopefully give insights into communications in more complex and varied biofilm communities.

Cystic fibrosis (CF) disease provokes the accumulation of thick and viscous sputum in the lungs, favoring the development of chronic and polymicrobial infections. Pseudomonas aeruginosa is the main bacterium responsible for these chronic infections, and much of the difficulty involved in eradicating it is due to biofilm formation. However, this could be mitigated using adjuvant compounds that help or potentiate the antibiotic action. Therefore, the main goal of this study was to search for substances that function as adjuvants and also as biofilm-controlling compounds, preventing or dismantling P. aeruginosa biofilms formed in an in vitro CF airway environment. Dual combinations of compounds with subinhibitory (1 and 2 mg/L) and inhibitory concentrations (4 mg/L) of ciprofloxacin were tested to inhibit the bacterial growth and biofilm formation (prophylactic approach) and to eradicate 24-h-old P. aeruginosa populations, including planktonic cells and biofilms (treatment approach). Our results revealed that aspartic acid (Asp) and succinic acid (Suc) restored ciprofloxacin action against P. aeruginosa. Suc combined with 2 mg/L of ciprofloxacin (Suc-Cip) was able to eradicate bacteria, and Asp combined with 4 mg/L of ciprofloxacin (AspCip) seemed to eradicate the whole 24-h-old populations, including planktonic cells and biofilms. Based on biomass depletion data, we noted that Asp induced cell death and Suc seemed somehow to block or reduce the expression of ciprofloxacin resistance. As far as we know, this kind of action had not been reported up till now. The presence of Staphylococcus aureus and Burkholderia cenocepacia did not affect the efficacy of the AspCip and SucCip therapies against P. aeruginosa and, also important, P. aeruginosa depletion from polymicrobial communities did not create a window of opportunity for these species to thrive. Rather the contrary, Asp and Suc also improved ciprofloxacin action against B. cenocepacia. Further studies on the cytotoxicity using lung epithelial cells indicated toxicity of SucCip caused by the Suc. In conclusion, we provided evidences that Asp and Suc could be potential ciprofloxacin adjuvants to eradicate P. aeruginosa living within polymicrobial communities. AspCip and SucCip could be promising therapeutic options to cope with CF treatment failures., This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020— Programa Operacional Regional do Norte. The authors acknowledge COMPETE2020 and FCT for the project POCI-01-0145-FEDER-029841 and for Scientific Employment Stimulus 2017 (CEECIND/01507/2017) provided to AS, info:eu-repo/semantics/publishedVersion

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioflm.2019.100010., The lack of reproducibility of published studies is one of the major issues facing the scientific community, and the field of biofilm microbiology has been no exception. One effective strategy against this multifaceted problem is the use of minimum information guidelines. This strategy provides a guide for authors and reviewers on the necessary information that a manuscript should include for the experiments in a study to be clearly interpreted and independently reproduced. As a result of several discussions between international groups working in the area of biofilms, we present a guideline for the spectrophotometric and fluorometric assessment of biofilm formation in microplates. This guideline has been divided into 5 main sections, each presenting a comprehensive set of recommendations. The intention of the minimum information guideline is to improve the quality of scientific communication that will augment interlaboratory reproducibility in biofilm microplate assays., This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska – Curie grant agreement No 722467, as part of the Print-Aid consortium. The information and views set out in this article are those of the authors and do not necessarily reflect the official opinion of the European Union. Neither the European Union institutions and bodies nor any person acting on their behalf may be held responsible for the use which may be made of the information contained therein. This work received additional financial support by: project UID/EQU/00511/2019 - Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE funded by national funds through FCT/MCTES (PIDDAC); Project “LEPABE-2-ECO-INNOVATION” – NORTE-01-0145-FEDER-000005, funded by Norte Portugal Regional Operational Programme (NORTE 2020), under PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)., info:eu-repo/semantics/publishedVersion

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request., Selecting appropriate tools providing reliable quantitative measures of individual populations in biofilms is critical as we now recognize their true polymicrobial and heterogeneous nature. Here, plate count, quantitative real-time polymerase chain reaction (q-PCR) and peptide nucleic acid probe-fluorescence in situ hybridization (PNA-FISH) were employed to quantitate cystic fibrosis multispecies biofilms. Growth of Pseudomonas aeruginosa, Inquilinus limosus and Dolosigranulum pigrum was assessed in dual- and triple-species consortia under oxygen and antibiotic stress. Quantification methods, that were previously optimized and validated in planktonic consortia, were not always in agreement when applied in multispecies biofilms. Discrepancies in culture and molecular outcomes were observed, particularly for triple-species consortia and antibiotic-stressed biofilms. Some differences were observed, such as the higher bacterial counts obtained by q-PCR and/or PNA-FISH (?4 log10 cells/cm2) compared to culture. But the discrepancies between PNA-FISH and q-PCR data (eg D. pigrum limited assessment by q-PCR) demonstrate the effect of biofilm heterogeneity in method's reliability. As the heterogeneity in biofilms is a reflection of a myriad of variables, tailoring an accurate picture of communities? changes is crucial. This work demonstrates that at least two, but preferentially three, quantification techniques are required to obtain reliable measures and take comprehensive analysis of polymicrobial biofilm-associated infections., The authors thank the financial support from the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI-01–0145-FEDER-006684) and BioTecNorte operation (NORTE01-0145-FEDER-000004) funded by the European Regional Development Fund (ERDF) under the scope of Norte2020 - Programa Operacional Regional do Norte. This work was also the result of the projects: (i) POCI01-0145-FEDER-006939 (Laboratory for Processing Engineering, Environment, Biotechnology and Energy – UID/EQU/00511/2013) funded by the ERDF, through COMPETE2020 and by national funds, through FCT; (ii) NORTE-01-0145-FEDER-000005 – LEPABE-2-ECO-INNOVATION, supported by North Portugal Regional Operational Programme (NORTE2020), under the Portugal2020 Partnership Agreement, through the ERDF; (iii) Coded-FISH PTDC/DTP-PIC/4562/2014/16678; (iv) POCI-01-0145-FEDER-029841, through COMPETE2020 - Programa Operacional Competitividade e Internacionalização and by national funds, through FCT). Also, the fellowship of Susana P. Lopes SFRH/BPD/95616/2013 is acknowledged. The authors would also like to thanks to Dr Michael Surette (Farncombe Family Digestive Health Research Institute, McMaster University, Hamilton, ON, Canada) for kindly providing the I. limosus strain used in this study., info:eu-repo/semantics/publishedVersion

Escherichia coli has developed sophisticated means to sense, respond, and adapt in stressed environment. It has served as a model organism for studies in molecular genetics and physiology since the 1960s. Stress response genes are induced whenever a cell needs to adapt and survive under unfavorable growth conditions. Two of the possible important genes are rpoS and bolA. The rpoS gene has been known as the alternative sigma () factor, which controls the expression of a large number of genes, which are involved in responses to various stress factors as well as transition to stationary phase from exponential form of growth. Morphogene bolA response to stressed environment leads to round morphology of E. coli cells, but little is known about its involvement in biofilms and its development or maintenance. This study has been undertaken to address the adherence pattern and formation of biofilms by E. coli on stainless steel, polypropylene, and silicone surfaces after 24 h of growth at 37 °C. Scanning electron microscopy was used for direct examination of the cell attachment and biofilm formation on various surfaces and it was found that, in the presence of bolA, E. coli cells were able to attach to the stainless steel and silicone very well. By contrast, polypropylene surface was not found to be attractive for E. coli cells. This indicates that bolA responded and can play a major role in the presence and absence of rpoS in cell attachment., info:eu-repo/semantics/publishedVersion

Worldwide, infections are resuming their role as highly effective killing diseases, as current treatments are failing to respond to the growing problem that is antimicrobial resistance (AMR). The social and economical burden of AMR seems ever rising, with health- and research-related organizations rushing to collaborate on a worldwide scale to find effective solutions. Resistant bacteria are spreading even in first-world nations, being found not only in healthcare-related settings, but also in food and in the environment. In this mini-review, the impact of AMR in healthcare systems and the major bacteria behind it are highlighted. Ecological aspects of AMR evolution and the complexity of its molecular mechanisms are explained. Major concepts, such as intrinsic, acquired, and adaptive resistance, as well as tolerance and heteroresistance, are also clarified. More importantly, the problematic of biofilms and their role in AMR, namely its main resistance and tolerance mechanisms, is elucidated. Finally, some of the most promising anti-biofilm strategies being investigated are reviewed. Much is still to be done regarding the study of AMR and the discovery of new anti-biofilm strategies. Gladly, considerable research on this topic is generated every day and increasingly concerted actions are being engaged globally to try and tackle this problem., This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors also acknowledge COMPETE2020 and FCT for the project POCI-01-0145-FEDER-029841, and FCT for the PhD Grants of Andreia Magalhães [grant number SFRH/BD/132165/2017] and Tânia Grainha [grant number SFRH/BD/136544/2018]., info:eu-repo/semantics/publishedVersion

Microbial colonisation of urinary catheters remains a serious problem for medicine as it often leads to biofilm formation and infection. Among the approaches reported to deal with this problem, surfaces functionalization to render them with antimicrobial characteristics, comprises the most promising one. Most of these strategies, however, are designed to target bacterial biofilms, while fungal biofilms are much less taken into account. In real-life settings, fungi will be inevitably found in consortium with bacteria, especially in the field of biomaterials. The development of antifungal coating strategies to be combined with antibacterial approaches will be pivotal for the fight of biomaterial-associated infections. The main goal of the present study was, therefore, to engineer an effective strategy for the immobilization of liposomal amphotericin B (LAmB) on polydimethylsiloxane (PDMS) surfaces to prevent Candida albicans colonization. Immobilization was performed using a two-step mussel-inspired coating strategy, in which PDMS are first immersed in dopamine solution. Its self-polymerization leads to the deposition of a thin adherent film, called polydopamine (pDA), which allowed the incorporation of LAmB, afterwards. Different concentrations of LAmB were screened in order to obtain a contact-killing surface with no release of LAmB. Surface characterization confirmed the polymerization of dopamine and further functionalization with LAmB yielded surfaces with less roughness and more hydrophilic features. The proposed coating strategy rendered the surfaces of PDMS with the ability to prevent the attachment of Candida albicans and kill the adherent cells, without toxicity towards mammalian cells. Overall results showed that LAmB immobilization on a surface retained its antifungal activity and reduced toxicity, holding therefore a great potential to be applied for the design of urinary catheters. Since the sessile communities commonly found associated to these devices exhibit a polymicrobial nature, the next challenge will be to co-immobilize LAmB with antibacterial agents to prevent the establishment of catheter urinary-associated infections., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors also acknowledge the support, through the Programa Operacional Competitividade e Internacionalizacao (COMPETE2020) and by national funds, through the Portuguese Foundation for Science and Technology (FCT), of the POLY-PrevEnTT project (PTDC/BTM-SAL/29841/2017-POCI-01-0145-FEDER-029841)., info:eu-repo/semantics/publishedVersion

These days, in hospital environment, reprocessing of Single-Use medical Devices (SUDs) is a practice used more and more often. Reprocessing of SUDs, in-house or by outsourcing, is a way for hospitals to produce cost savings and less quantity of waste. This review focuses on the evolution of SUDs reprocessing, as well as on the legislation and regulation associated, and discusses the potential risks involved. Historical evolution unveils that reprocessing is unequal between developed and developing countries, being the United States and Canada the more reprocessors countries. The control of this service ensures the safety of this practice, as it happens in developed countries. It is expected that the market of reprocessed SUDs will grow, and more hospitals will begin to reprocess these devices., (undefined), info:eu-repo/semantics/publishedVersion

Pseudomonas aeruginosa chronic infections are the major cause of high morbidity and mortality in cystic fibrosis (CF) patients due to the use of sophisticated mechanisms of adaptation, including clonal diversification into specialized CF-adapted phenotypes. In contrast to chronic infections, very little is known about what occurs after CF lungs colonization and at early infection stages. This study aims to investigate the early events of P. aeruginosa adaptation to CF environment, in particular, to inspect the occurrence of clonal diversification at early stages of infection development and its impact on antibiotherapy effectiveness. To mimic CF early infections, three P. aeruginosa strains were long-term grown in artificial sputum (ASM) over 10 days and phenotypic diversity verified through colony morphology characterization. Biofilm sub- and inhibitory concentrations of ciprofloxacin were applied to non- and diversified populations to evaluate antibiotic effectiveness on P. aeruginosa eradication. Our results demonstrated that clonal diversification might occur after ASM colonization and growth. However, this phenotypic diversification did not compromise ciprofloxacin efficacy in P. aeruginosa eradication since a biofilm minimal inhibitory dosage would be applied. The expected absence of mutators in P. aeruginosa populations led us to speculate that clonal diversification in the absence of ciprofloxacin treatments could to be driven by niche specialization. Yet, biofilm sub-inhibitory concentrations of ciprofloxacin seemed to overlap niche specialization as fitter variants had emerged, such as mucoid, small colony and pinpoint variants, known to be highly resistant to antibiotics. The pathogenic potential of all emergent colony morphotypes-associated bacteria, distinct from the wild-morphotypes, revealed that P. aeruginosa evolves to a non-swimming phenotype. Impaired swimming motility seemed to be one of the first evolutionary steps of P. aeruginosa in CF lungs that could pave the way for further adaptation steps including biofilm formation and progress to chronic infection. Based on our findings, impaired swimming motility seemed to be a candidate to disease marker of P. aeruginosa infection development. Despite our in vitro CF model represents a step forward towards in vivo scenario and it provided valuable insights about the early events, more and distinct P. aeruginosa strains should be studied to strengthen our results., This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/ BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors also acknowledge COMPETE2020 and FCT for the project POCI-01-0145FEDER-029841., info:eu-repo/semantics/publishedVersion

The combination of antimicrobial products is a promising biomedical strategy against the ever growing number of resistant strains emerging in healthcare and community settings. Agents with alternative modes of action, such as antimicrobial peptides (AMPs), and the efficacy of combined actions are being evaluated. Despite the availability of various antimicrobial data repositories, a wealth of information remains scattered through the scientific literature. This work introduces the automated curation pipeline implemented to produce the first ever network reconstruction of AMP-drug combinations. This network relates to antimicrobial combinations experimentally tested against Pseudomonas aeruginosa infections and includes 239 combinations among AMPs and other antimicrobials. The aim is to provide a global view of available interaction data and help design new antimicrobial studies. Reconstruction is on-going, coping with new experimental results for P. aeruginosa, and will be soon extended to other meaningful microbial pathogens. The network is publicly accessible at http://sing.ei.uvigo.es/antimicrobialCombination/., The research leading to these results has received funding from: the European Union's Seventh Framework Programme FP7/REGPOT-2012-2013.1 under grant agreement n° 316265, BIOCAPS; FCT through the project AntiPep PTDC/SAU-SAP/113196/2009/FCOMP-01-0124-FEDER016012; the Strategic Project PEst-OE/EQB/LA0023/2013; the Project “BioHealth - Biotechnology and Bioengineering approaches to improve health quality", NORTE-07-0124FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER; the project “RECI/BBB-EBI/0179/2012 - Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB”, FCOMP-01-0124-FEDER027462; and the Agrupamento INBIOMED from DXPCTSUG-FEDER unha maneira de facer Europa (2012/273). This document reflects only the author’s views and the European Union is not liable for any use that may be made of the information contained herein. The authors also acknowledge the Foundation for Science and Technology (FCT) for the PhD Grant of Paula Jorge, Ref. SFRH/BD/88192/2012. The authors do not have any conflicts of interest., info:eu-repo/semantics/publishedVersion

Contract grant sponsor: FCT, COMPETE 2020, FEDER, PT2020Contract grant number: UID/BIO/04469/2013Contract grant sponsor: FCT/MEC, FEDER, PT2020Contract grant number: UID/EQU/00511/2013-LEPABEContract grant sponsor: COMPETE 2020Contract grant number: POCI-01-0145-FEDER-006684Contract grant sponsor: FCTContract grant number: RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462)Contract grant sponsor: QREN, FEDER, ON2Contract grant number: NORTE-07-0124-FEDER-000027Contract grant sponsor: FEDER, ON2, FCT/MECContract grant number: NORTE-07-0124-FEDER-000025Contract grant sponsor: COST-Action TD1004

Biofilms plays an important role in medical-device-related infections. This study aimed to determine the factors that influence adherence and biofilm production, as well as the relationship between strong biofilm production and genetic determinants in clinical isolates of meticillin-resistant Staphylococcus aureus (MRSA). Fifteen strains carrying different chromosomal cassettes recovered from hospitalized patients were selected; five SCCmecII, five SCCmecIII and five SCCmecIV. The SCCmec type, agr group and the presence of the virulence genes (bbp, clfA, icaA, icaD, fnbB, bap, sasC and IS256) were assessed by PCR. PFGE and multilocus sequence typing (MLST) techniques were also performed. The initial adhesion and biofilm formation were examined by quantitative assays. The surface tension and hydrophobicity of the strains were measured by the contact angle technique to evaluate the association between these parameters and adhesion ability. SCCmecIII and IV strains were less hydrophilic, with a high value for the electron acceptor parameter and higher adhesion in comparison with SCCmecII strains. Only SCCmecIII strains could be characterized as strong biofilm producers. The PFGE showed five major pulsotypes (AE); however, biofilm production was related to the dissemination of one specific PFGE clone (C) belonging to MLST ST239 (Brazilian epidemic clonal complex). The genes agrI, fnbB and IS256 in SCCmecIII strains were considered as genetic determinants associated with strong biofilm-formation by an ica-independent biofilm pathway. This study contributes to the understanding of biofilm production as an aggravating factor potentially involved in the persistence and severity of infections caused by multidrug-resistant MRSA belonging to this genotype., We thank FAPEMIG (Fundação de Amparo à Pesquisa de Minas Gerais, proceeding APQ 01398-11) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, PDSE proceeding 8952/11-6) for the financial support and scholarships. We also thank Dr Teruyo Ito, Juntendo University, Japan, and Dr Elsa Masae Mamizuka, Universidade de São Paulo, Brazil, for kindly providing the control strains used in this study., info:eu-repo/semantics/publishedVersion

Biofilm research is growing more diverse and dependent on high-throughput technologies and the large-scale production of results aggravates data substantiation. In particular, it is often the case that experimental protocols are adapted to meet the needs of a particular laboratory and no statistical validation of the modified method is provided. This paper discusses the impact of intra-laboratory adaptation and non-rigorous documentation of experimental protocols on biofilm data interchange and validation. The case study is a non-standard, but widely used, workflow for Pseudomonas aeruginosa biofilm development, considering three analysis assays: the crystal violet (CV) assay for biomass quantification, the XTT assay for respiratory activity assessment, and the colony forming units (CFU) assay for determination of cell viability. The ruggedness of the protocol was assessed by introducing small changes in the biofilm growth conditions, which simulate minor protocol adaptations and non-rigorous protocol documentation. Results show that even minor variations in the biofilm growth conditions may affect the results considerably, and that the biofilm analysis assays lack repeatability. Intra-laboratory validation of non-standard protocols is found critical to ensure data quality and enable the comparison of results within and among laboratories., We thank the Portuguese Foundation for Science and Technology (FCT) the strategic funding of UID/BIO/04469/2013 unit. The authors also acknowledge the support by FCT and the European Community fund FEDER, through Program COMPETE, under the scope of the Projects AntiPep PTDC/SAU-SAP/113196/2009 (FCOMP-01-0124-FEDER-016012), RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), the Projects "BioHealth-Biotechnology and Bioengineering approaches to improve health quality," NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER, and the Agrupamento INBIOMED from DXPCTSUG-FEDER unha maneira de facer Europa (2012/273). The research leading to these results has received funding from the European Union's Seventh Framework Programme FP7/REGPOT-2012-2013.1 under grant agreement No. 316265, BIOCAPS. This document reflects only the author's views, and the European Union is not liable for any use that may be made of the information contained herein. The authors also acknowledge the Ph.D. Grant of Paula Jorge, Ref. SFRH/BD/88192/2012.

Since most antibacterial coatings reported to fight biomaterial-associated infections (BAI) fail in completely preventing bacterial colonization, it is crucial to know the impact of that small fraction of adhered bacteria in BAI recrudescence. This study aims to understand the fate of Staphylococcus aureus able to adhere to an antimicrobial coating previously developed, in terms of potential development of bacterial resistance and their macrophage-mediated phagocytosis. Antimicrobial coating comprised the co-immobilization of Palm peptide and DNase I onto polydimethylsiloxane. Expression of genes associated to resistance and virulence mechanisms showed that cells in contact with antimicrobial surfaces for a long period of 30 days, exhibit genes equally or less expressed, as compared to cells recovered from control surfaces. Recovered cells also exhibit the same susceptibility patterns, which strengthens the evidence of no resistance development. Remarkably, cells adhered to modified surfaces shows a reduced metabolic activity upon vancomycin treatment unlike the cells found on control surfaces, which can be identified as a clinical opportunity for prophylactically administration after implant surgery. Furthermore, results highlight that functionalization of PDMS with Palm and DNase I should not compromise the action of host immune cells. The overall results reinforce the potential of this antimicrobial strategy to fight BAI., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01 0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. The authors also acknowledge the support by FCT and the European Community fund FEDER, through Program COMPETE, under the scope of the Project AntiPep PTDC/SAUSAP/113196/2009 (FCOMP-01-0124-FEDER-016012) and the PhD Grant of Diana Alves (SFRH/BD/78063/2011) and Andreia Magalhães (SFRH/BD/132165/2017). A special thanks to Doctor Agostinho Carvalho and Doctor Cristina Amorim from Life and Health Sciences Research Institute (ICVS), University of Minho for kindly providing the monocyte cell line used in this study. Doctor Nuno Cerca, from CEB, Centre of Biological Engineering, University of Minho, is also acknowledged for his important contribution on the interpretation of gene expression results., info:eu-repo/semantics/publishedVersion

The complex virulence attributes of Candida albicans are an attractive target to exploit in the development of new antifungals and anti-virulence strategies to combat C. albicans infections. Particularly, quorum-sensing (QS) has been reported as critical for virulence regulation in C. albicans. This work presents two knowledge networks with up-to-date information about QS regulation and experimentally tested anti-QS and anti-virulence agents for C. albicans. A semi-automatic bioinformatics workflow that combines literature mining and expert curation was used to retrieve otherwise scattered information from the scientific literature. The network representation offers an innovative and continuously updatable means for the Candida research community to query QS and virulence data systematically and in a user-friendly way. Notably, the reconstructed networks show the complexity of QS regulation and the impact that some molecules have on the inhibition of virulence mechanisms responsible for infection establishment (e.g. hyphal development) and perseverance (e.g. biofilm formation). In the future, the compiled knowledge may be used to build decision-making models that help infer new knowledge of practical significance. The knowledge networks are publicly available at http://pcquorum.org/. This Web platform enables the exploration of fungal virulence cues as well as reported inhibitors in a user-friendly fashion., This work was supported by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) [Research Grant 2014 of Analia Lourenc¸o], the Portuguese Foundation for Sci- ´ ence and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI01-0145-FEDER-006684) and BioTecNorte operation ( NORTE01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. MP and GPR were supported by pre-doctoral fellowships from the Xunta de Galicia., info:eu-repo/semantics/publishedVersion

Summary Biofilm studies are at the crossroads of Biology, Chemistry, Medicine, Material Science and Engineering, among other fields. Data harmonisation in Biofilms is therefore crucial to allow for researchers to collaborate, interchange, understand, and replicate studies at an inter-laboratory and inter-domain scale. The international Minimum Information About a Biofilms Experiment initiative has prepared a set of guidelines for documenting biofilms experiments and data, namely the minimum information checklist. This paper goes a step forward and describes a new ontology for the broad description of biofilm experiments and data. In such an interdisciplinary context we chose to rely on a common integration framework provided by a foundational ontology that facilitates the addition and extension of various sub-domain modules, and the consistent integration of terminology extracted from several existing ontologies, e.g. EXPO and ChEBI. The community is participating actively in the production of this resource, and it is already used by public biofilms-centred databases, such as BiofOmics, and bioinformatics tools, such as the Biofilms Experiment Workbench. This practical validation serves the purpose of disseminating the controlled vocabulary among researchers and identifying current limitations, glitches, and inconsistencies. Information branches will be added, extended or refactored according to user feedback and group discussions.

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ijantimicag.2017.02.012., Antimicrobial combinations involving antimicrobial peptides (AMPs) attract considerable attention within current antimicrobial and anti-resistance research. The objective of this study was to review the available scientific literature on the effects of antimicrobial combinations involving colistin (polymyxin E), polymyxin B and nisin, which are US Food and Drug Administration (FDA)-approved AMPs broadly tested against prominent multidrug-resistant pathogens. A bioinformatics approach based on literature mining and manual expert curation supported the reconstruction of experimental evidence on the potential of these AMP combinations, as described in the literature. Network analysis enabled further characterisation of the retrieved antimicrobial agents, targets and combinatory effects. This systematic analysis was able to output valuable information on the studies conducted on colistin, polymyxin B and nisin combinations. The reconstructed networks enable the traversal and browsing of a large number of agent combinations, providing comprehensive details on the organisms, modes of growth and methodologies used in the studies. Therefore, network analysis enables a bird's-eye view of current research trends as well as in-depth analysis of specific drugs, organisms and combinatory effects, according to particular user interests. The reconstructed knowledge networks are publicly accessible at http://sing-group.org/antimicrobialCombination/. Hopefully, this resource will help researchers to look into antimicrobial combinations more easily and systematically. User-customised queries may help to identify missing and less studied links and to generate new research hypotheses., This work was supported by the Portuguese Foundation for Science and Technology(FCT)under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 [POCI01-0145-FEDER-006684] and BioTecNorte operation [NORTE-010145-FEDER-000004], funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte.The authors also acknowledge the support received from FCT and the European Community fund FEDER, through Program COMPETE, under the scope of the Project RECI/BBB-EBI/0179/2012 [FCOMP-01-0124-FEDER-027462],the[14VI05]Contract-Programme from the University of Vigo (Vigo, Spain), the INOU-16-05 project from the Provincial Council of Ourense, and the Agrupamento INBIOMED from DXPCTSUG-FEDER unha maneira de facer Europa [2012/273]. SING group thanks CITI (Centro de Investigación, Transferencia e Innovación) from University of Vigo for hosting its IT infrastructure. Finally, the authors acknowledge the PhD grant of Paula Jorge[Grant no. SFRH/BD/88192/2012],funded by FCT,thePhD grants of Martín Pérez-Pérez and Gael Pérez-Rodríguez, funded by the Xunta de Galicia and the University of Vigo, and the Research grant 2014 of Anália Lourenço by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID)., info:eu-repo/semantics/publishedVersion

Antimicrobial research is being pressured to look for more effective therapeutics for the ever-growing antibiotic-resistant infections, and antimicrobial peptides (AMP) and antimicrobial combinations are promising solutions. This work evaluates colistin-AMP combinations against two major pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, encompassing non- and resistant strains. Colistin (CST) combined with the AMP temporin A (TEMP-A), citropin 1.1 (CIT-1.1) and tachyplesin I linear analogue (TP-I-L) was tested against planktonic, single- and double-species biofilm cultures. Overall synergy for planktonic P. aeruginosa and synergy/additiveness for planktonic S. aureus were observed. Biofilm growth prevention was achieved with synergy and additiveness. Pre-established 24 h-old biofilms were harder to eradicate, especially for S. aureus and double-species biofilms; still, some synergy and addictiveness was observed for higher concentrations, including for the biofilms of resistant strains. Different treatment times and growth media did not greatly influence AMP activity. CST revealed low toxicity compared with the other AMP but its combinations were toxic for high concentrations. Overall, combinations reduced effective AMP concentrations, mainly in prevention scenarios. Improvement of effectiveness and toxicity of therapeutic strategies will be further investigated., The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) (http://www.fct.pt/), under the scope of the strategic funding of UID/B10/04469/2013 and COMPETE 2020 (POCI-01-0145-FEDER-006684). This study was also supported by FCT and the European Community fund FEDER, through Program COMPETE, and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 -Programa Operacional Regional do Norte. This work was also partially funded by the [14V105] Contract-Programme from the University of Vigo (https://mw.uvigo.gal/ uvigo_en/) and the Agrupamento INBIOMED (http://inbiomed.webs.uvigaes/) from DXPCTSUG-FEDER unha maneira de facer Europa (2012/273) and co-financed by the European Regional Development Fund (http://ec.europleuiregionaL policy/EN/fundingierdf/) under the Operational Programme Innovative Economy (WNP-POIG.01.04.00-22-052/11).). Lipopharm.pl (http://www.lipopharm.p1/) provided support in the form of salaries for authors DG and WK. The authors also acknowledge the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) (https://www.escmid.org/) for the Research Grant 2014 to Anglia Lourenco, and FCT for the PhD Grant of Paula Jorge (grant number SFRH/BD/88192/2012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., info:eu-repo/semantics/publishedVersion

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.02146/full#supplementary-material, Cystic Fibrosis (CF) airways disease involves complex polymicrobial infections where different bacterial species can interact and influence each other and/or even interfere with the whole community. To gain insights into the role that interactions between Pseudomonas aeruginosa in co-culture with Staphylococcus aureus, Inquilinus limosus,and Stenotrophomonas maltophilia may play in infection, the reciprocal effect during biofilm formation and the response of dual biofilms toward ciprofloxacin under in vitro atmospheres with different oxygen availabilities were evaluated. Biofilm formation kinetics showed that the growth of S. aureus, I. limosus, and S. maltophilia was disturbed in the presence of P. aeruginosa, under both aerobic and anaerobic environments. On the other hand, under aerobic conditions, I. limosus led to a decrease in biofilm mass production by P. aeruginosa, although biofilm-cells viability remains unaltered. The interaction between S. maltophilia and P. aeruginosa positively influenced dual biofilm development by increasing its biomass. Compared with monocultures, biomass of P. aeruginosaC S. aureus biofilms was significantly reduced by reciprocal interference. When grown in dual biofilms with P. aeruginosa, ciprofloxacin was less effective against S. aureus, I. limosus, and S. maltophilia, with increasing antibiotic doses leading to drastic inhibitions of P. aeruginosa cultivability. Therefore, P. aeruginosa might be responsible for the protection of the whole dual consortia against ciprofloxacin activity. Based on the overall data, it can be speculated that reciprocal interferences occur between the different bacterial species in CF lung, regardless the level of oxygen. The findings also suggest that alterations of bacterial behavior due to species interplay may be important for disease progression in CF infection., The authors acknowledge the Portuguese Foundation for Science and Technology (FCT), under the scope of the strategic funding of UID/BIO/04469/2013 and COMPETE 2020 (POCI01-0145-FEDER-006684). This study was also supported by FCT and the European Community fund FEDER, through Program COMPETE, under the scope of the Projects RECI/BBBEBI/0179/2012 (FCOMP-01-0124-FEDER-027462), “BioHealth– Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027 and NORTE-070124-FEDER-000025 – RL2_ Environment & Health, co-funded by the Programa Operacional Regional do Norte (ON.2 – O NovoNorte), QREN, FEDER. The authors also acknowledge the grants of Susana P. Lopes (SFRH/BPD/95616/2013) and Andreia P.Magalhães (UMINHO/BD/25/2016).

Cystic fibrosis (CF) infections are invariably biofilm-mediated and polymicrobial, being safe to assume that a myriad of factors affects the sociomicrobiology within the CF infection site and modulate the CF community dynamics, by shaping their social activities, overall functions, virulence, ultimately affecting disease outcome. This work aimed to assess changes in the dynamics (particularly on the microbial composition) of dual-/three-species biofilms involving CF-classical (Pseudomonas aeruginosa) and unusual species (Inquilinus limosus and Dolosigranulum pigrum), according to variable oxygen conditions and antibiotic exposure. Low fluctuations in biofilm compositions were observed across distinct oxygen environments, with dual-species biofilms exhibiting similar relative proportions and P. aeruginosa and/or D. pigrum populations dominating three-species consortia. Once exposed to antibiotics, biofilms displayed high resistance profiles, and microbial compositions, distributions, and microbial interactions significantly challenged. The antibiotic/oxygen environment supported such fluctuations, which enhanced for three-species communities. In conclusion, antibiotic therapy hugely disturbed CF communities dynamics, inducing significant compositional changes on multispecies consortia. Clearly, multiple perturbations may disturb this dynamic, giving rise to various microbiological scenarios in vivo, and affecting disease phenotype. Therefore, an appreciation of the ecological/evolutionary nature within CF communities will be useful for the optimal use of current therapies and for newer breakthroughs on CF antibiotherapy., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/ BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684). The authors also acknowledge the financial support provided by FCT through the projects: PTDC/SAU-ESA/646091/2006/ FCOMP01-0124-FEDER-007480FCT; strategic project PEst-OE/EQB/LA0023/2013; “BioHealth – Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027, cofunded by the Programa Operacional Regional do Norte (ON.2–O Novo Norte), QREN, FEDER; RECI/BBB-EBI/0179/2012 – Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB, FCOMP-01-0124-FEDER-027462, FEDER; and the DNA mimics project PIC/IC/82815/2007. The FCT BPD fellowship of Susana P. Lopes SFRH/BPD/95616/2013 and the support of the COST-Action TD1004:Theragnostics for imaging and therapy is also acknowledged., info:eu-repo/semantics/publishedVersion

This study aimed to evaluate the effect of the compound 1,4-dihydroxy-2E,6E,12E-trien-5-one-casbane (CD1CN), a casbane diterpene isolated from Croton nepetaefolius stalks, on the biofilm formation or preformed biofilms of bacteria and yeasts. Minimum inhibitory concentration results showed that CD1CN inhibited the growth of single cultures of Staphylococcus aureus and Candida albicans at 125 and 500 g/mL, respectively, as well as dual cultures of S. aureus with C. albicans or Candida glabrata at 500 and 250 g/mL, respectively. In general, CD1CN reduced biofilm biomass when applied to preformed biofilms or when applied during the biofilm formation of single and dual cultures in concentrations ranging from 31.25 to 250 g/mL, depending on the culture. CD1CN was more effective in reducing the cfu of S. aureus in single and dual biofilms (62.5–250 g/mL) than that of Pseudomonas aeruginosa, although this reduction was also significant. For yeasts, CD1CN was generally more effective in reducing C. glabrata cfu in single or dual cultures when compared to C. albicans. SEM images of the dual-species biofilms confirmed these results. In conclusion, CD1CN could be an effective alternative to conventional antimicrobial agents against infectious biofilms, in particular those attributed to mixed cultures., This study was supported by the Coordenacao de Aperfeicomento de Pessoal de Nivel Superior (CAPES - Brazil) through the BEX NT 2052/11NT3 project managed by the Instituto de Biotecnologia e Bioengenharia-Centro de Engenharia Biologica (IBB-CEB) and Fundacao para a Ciencia e a Tecnologia (FCT - Portugal); by the European Community fund FEDER through the COMPETE Program under the auspices of Project PTDC/SAU-ESA/646091/2006/FCOMP-01-0124-FEDER-007480; and by Fundacao Cearense de Apoio ao Desenvolvimento Cientifico e Tecnologico (Funcap - Brazil) through the BPI-0067-00068.01.00/12 project. BSC and EHT are senior researchers of CNPq. Mr. David Martin helped with the English editing of the manuscript that was also revised by AJE (American Journal Experts).

Pseudomonas aeruginosa is the most prevalent pathogen of cystic fibrosis (CF) lung disease. Its long persistence in CF airways is associated with sophisticated mechanisms of adaptation, including biofilm formation, resistance to antibiotics, hypermutability and customized pathogenicity in which virulence factors are expressed according the infection stage. CF adaptation is triggered by high selective pressure of inflamed CF lungs and by antibiotic treatments. Bacteria undergo genetic, phenotypic, and physiological variations that are fastened by the repeating interplay of mutation and selection. During CF infection development, P. aeruginosa gradually shifts from an acute virulent pathogen of early infection to a host-adapted pathogen of chronic infection. This paper reviews the most common changes undergone by P. aeruginosa at each stage of infection development in CF lungs. The comprehensive understanding of the adaptation process of P. aeruginosa may help to design more effective antimicrobial treatments and to identify new targets for future drugs to prevent the progression of infection to chronic stages., The authors thank the project FCT PTDC/SAUSAP/113196/2009/FCOMP-01-0124-FEDER-016012, the Strategic Project PEst-OE/EQB/LA0023/2013, the Project. BioHealth-Biotechnology and Bioengineering approaches to improve health quality., Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER, the project. RECI/BBB-EBI/0179/2012-Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB., Ref. FCOMP-01-0124-FEDER-027462, FEDER. The authors also acknowledge PhD Grant of Ana MargaridaSousa SFRH/BD/72551/2010.

Aim To evaluate the synergistic activity of antimicrobial drugs against lineages of methicillin-resistant Staphylococcus aureus (MRSA) carrying SCCmec IV. The biofilm production and related genes were also detected. Methods and Results Forty two MRSA isolates were tested for biofilm production and related genes. Biofilm/biomass susceptibility to gentamicin (G), linezolid (L), rifampicin (R) and vancomycin (V) was determined for six isolates from three lineages prevalent in Rio de Janeiro hospitals in concentrations ranging from 0·25 to 64 μg ml−1. Biomass was evaluated by microtitre plate test and number of viable cells (CFU cm−2) and inspected by epifluorescence microscopy. All isolates presented the icaA and sasG genes, but only 38% were biofilm producers. There were 50 and 45% biomass reductions when concentrations ≥4 μg ml−1 of R or L and ≥16 μg ml−1 of G or V, respectively, were used. Synergism tests produced a 55% biomass reduction with R2lg ml1 + G16lg ml1 , R2lg ml1 + L2lg ml1 , R2lg ml1 + V4lg ml1 , and L2lg ml1 + V4lg ml1 . Number of viable cells was reduced from 2 to 3 logs with R2lg ml1 + L2lg ml1 and R2lg ml1 + V4lg ml1 . Conclusions Synergisms involving R plus L and R plus V caused important reductions in biofilm/biomass and the number of viable cells. Drug combinations should be considered in the chemotherapies of MRSA-SCCmec IV infections. Significance and Impact of the Study Biofilms in MRSA infections restrict the clinical choice of antimicrobials. Thus, knowledge of the best options for monotherapy and drug synergisms could improve clinical results., This study was supported by grants from Fundacao Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Coordenacao de Aperfeicoamento Pessoal de Nivel Superior (CAPES), Fundacao Universitaria Jose Bonifacio (FUJB) and Programa de Nucleos de Excelencia (PRONEX). The financial support through the projects: PTDC/SAUSAP/113196/2009/ FCOMP-01-0124-FEDER-016012; PEst-OE/EQB/LA0023/2013; 'BioHealth-Biotechnology and Bioengineering approaches to improve health quality', NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte, QREN; RECI/BBB-EBI/0179/2012/FCOMP-01-0124-FEDER-027462.

The minimum information about a biofilm experiment (MIABiE) initiative has arisen from the need to find an adequate and scientifically sound way to control the quality of the documentation accompanying the public deposition of biofilm-related data, particularly those obtained using high-throughput devices and techniques. Thereby, the MIABiE consortium has initiated the identification and organization of a set of modules containing the minimum information that needs to be reported to guarantee the interpretability and independent verification of experimental results and their integration with knowledge coming from other fields. MIABiE does not intend to propose specific standards on how biofilms experiments should be performed, because it is acknowledged that specific research questions require specific conditions which may deviate from any standardization. Instead, MIABiE presents guidelines about the data to be recorded and published in order for the procedure and results to be easily and unequivocally interpreted and reproduced. Overall, MIABiE opens up the discussion about a number of particular areas of interest and attempts to achieve a broad consensus about which biofilm data and metadata should be reported in scientific journals in a systematic, rigorous and understandable manner., The authors would like to thank Thomas Bjarnsholt and Adyary Fallarero for a critical revision of the manuscript. This work was supported by IBB-CEB; Fundacao para a Ciencia e Tecnologia (FCT); the European Community fund FEDER, through Program COMPETE, in the ambit of the FCT Project PTDC/SAU-SAP/113196/2009/FCOMP-01-0124-FEDER-0 16012; the European Union Seventh Framework Programme [FP7/REGPOT-2012-2013.1] under Grant Agreement No. 316265, BIOCAPS; the Agrupamento INBIOMED from DXPCTSUG-FEDER unha maneira de facer Europa (2012/273). T. C. would like to thank FWO Vlaanderen and the Interuniversity Attraction Poles Programme initiated by the Belgian Science Policy Office for funding.